Microgel culture in stirred tank reactors: Effects on extracellular vesicle secretion and mesenchymal stem cell function

Three-dimensional (3D) dynamic conditions are necessary for physiologically relevant mesenchymal stem cell (MSC) culture. Furthermore, bioreactor-based processes and hydrogel-encapsulation substantially improve niche standardization, expansion control, and process scalability. The integration of these technologies can overcome some challenges that prevent the clinical translation of MSCs. However, few studies have investigated the synergistic use of bioreactors and hydrogels, and none have explored the combination of high-throughput encapsulation platforms, such as microfluidics or millifluidics-generated microgels, with standard stirred-tank systems. In this study, we characterized the continuous culture of MSCs in a stirred-tank reactor, encapsulating the cells in gelatin methacryloyl (GelMA) microgels using a low-cost, user-friendly approach. The effects of seeding density, GelMA's degree of functionalization (DoF), bioreactor sampling protocol, and donor screening were assessed using cell metabolic activity, differentiation, proliferation, and extracellular vesicle (EV) secretion. Gentle dynamic culture enhanced MSCs' metabolic activity. However, cell proliferation was inhibited within the microgels, and no cell migration was observed on the hydrogel's surface. GelMA with a low DoF and high cell seeding density favored cell survival during culture, whereas pronounced donor-dependent differences were observed in cell proliferation and metabolism. The yield, size distribution, and protein content of MSC-EVs were affected by seeding density under dynamic 3D conditions. Furthermore, MSC differentiation led to readily measurable changes in the microgels. Our findings highlight the platform's potential for high throughput microtissue generation and efficient assessment of bioactive compounds.